骨软骨组织工程支架的研究现状及发展趋势

目前,随着骨软骨组织工程的发展,为临床上骨软骨缺损的修复带来了新希望。应用自体细胞、支架、生长因子可以修复骨、软骨的缺损;选取具有生物相容性和可吸收性的复合支架可为细胞提供暂时的支持、黏附、生长环境,促进骨软骨缺损的修复。就骨软骨组织工程支架的分类、特性、应用以及存在的问题和发展趋势作一综述。     

Regulation of mu opioid receptor internalization by the scaffold protein RanBPM

Mu opioid receptors (MOP) are transducers of the pharmacological effects of many opioid drugs, including analgesia and tolerance/dependence. Previously, we observed increased MOP signaling during postnatal development that was not associated with increased MOP or G protein expression. A yeast two-hybrid screen of a human brain cDNA library using the MOP C-terminus as bait identified RanBPM as a potential MOP-interacting protein. RanBPM has been recognized as a multi-functional scaffold protein that interacts with a variety of signaling receptors/proteins. Co-immunoprecipitation studies in HEK293 cells indicated that RanBPM constitutively associates with MOP. Functionally, RanBPM had no effect on MOP-mediated inhibition of adenylyl cyclase, yet reduced agonist-induced endocytosis of MOP. Mechanistically, RanBPM interfered with β arrestin2-GFP translocation stimulated by MOP but not α_1B-adrenergic receptor activation, indicating selectivity of action. Our findings suggest that RanBPM is a novel MOP-interacting protein that negatively regulates receptor internalization without altering MOP signaling through adenylyl cyclase.     

Development of hyaluronic acid-based scaffolds for brain tissue engineering

Three-dimensional biodegradable porous scaffolds play vital roles in tissue engineering. In this study, a hyaluronic acid–collagen (HA–Coll) sponge with an open porous structure and mechanical behavior comparable to brain tissue was developed. HA–Coll scaffolds with different mixing ratios were prepared by a freeze–drying technique and crosslinked with water-soluble carbodiimide to improve mechanical stability. The pore structure of the samples was evaluated by light and scanning electron microscopy, and the mechanical behavior was analyzed by mechanical compression and tension testing. The degree of crosslinking was determined by the water absorption and trinitrobenzene sulfonic assay, and the HA content was determined by a carbazole assay. The results showed that HA–Coll scaffolds containing an open porous structure with a homogeneous pore size distribution could be fabricated. Certain features of the mechanical properties of HA–Coll scaffolds prepared with a Coll:HA mixing ratio of 1:2, and pure HA sponges, were comparable with brain tissue. Neural stem cells (NSCs) were expanded in number in monolayer culture and then seeded onto the three-dimensional scaffolds in order to investigate the effects of the different types of scaffolds on neurogenic induction of the cells. This study contributes to the understanding of the effects of HA content and crosslink treatment on pore characteristics, and mechanical behavior essential for the design of HA–Coll scaffolds suitable for NSC growth and differentiation for brain tissue engineering.     

仿生组装纳米羟基磷灰石/壳聚糖骨修复材料的制备及其生物相容性研究

目的 探讨以一种简单、廉价的方法制备纳米羟基磷灰石/壳聚糖(n-HA/CS)复合材料,并评价其理化特征和生物相容性. 方法采用原位沉析和冷冻干燥法制备n-HA/CS支架,通过扫描电镜、组织切片染色、X线衍射和傅立叶红外光谱分析其微观形貌和组成;采用万能材料试验机分析材料的力学性能.采用材料浸提液和表面接种考察n-HA/CS复合材料对第3代人骨髓基质干细胞(hBMSCs)黏附、增殖的影响,评估其细胞相容性.将n-HA/CS复合材料植入新西兰大白兔背部肌袋,经组织学染色后评价其组织相容性. 结果 n-HA/CS复合材料具有多孔结构,孔隙率为(88.65±2.34)%,孔径为(112.63±20.47) μm,HA晶体颗粒长度为200～700 nm,且分散均匀;X线衍射和红外光谱分析表明合成的HA是含CO32-弱结晶纳米晶体.材料的断裂强度为(1.47±0.15)MPa,弹性模量为(37.52±3.43)kPa,可满足非负重部位骨修复要求.n-HA/CS材料浸提液未明显抑制hBMSCs的增殖,直接接种在n-HA/CS复合材料表面的细胞黏附、增殖功能正常;组织相容性实验也表明,植入4周后组织炎性反应明显减轻,12周后材料基本降解并由新生组织爬行替代. 结论采用原位沉析和冷冻干燥法制备的n-HA/CS复合材料具有良好的理化性质和生物相容性,有望应用于组织工程骨的构建.     

新型骨组织工程β-TCP／CPPF／PLLA支架复合BMSCs修复节段性骨缺损的影像学研究

目的：将自体骨髓基质细胞（Bone marrow stromal cells,BMSCs）接种到β-TCP／CPPF／PLLA支架上,进行体外复合培养,构建组织工程人工骨,植入兔桡骨大段骨缺损模型中,评估BMSCs／β-TCP／CPPF／PLLA复合体促进成骨的作用。方法：用新西兰大白兔40只,随机分为BMSCs／β-TCP／CPPF,PLL复合物组（A组）、β-TCP／CPPF／PLLA支架组（B组）、空白对照组（C组）共三组,建立兔桡骨双侧大段骨缺损模型,相应植入自体细胞材料复合物和单纯支架材料,空白对照组不作任何处理。术后4、8、12、16周处死动物,摄X线片观察骨缺损修复情况。结果：X线检查结果：A组术后2周可见缺损处有散在的、少量的模糊状骨痂生成,术后4周可见明显的骨生成影像,成云雾状,均匀分布在骨缺损区,术后8周整个缺损区均可见到骨痂生成,成骨现象较4周更加明显,部分髓腔已通,术后12～16周,缺损区已完全为新生的骨组织充填,骨髓腔已完全再通,修复区比正常的桡骨较细。B组、C组术后2～16周,虽有不同程度的成骨现象,但没有完全修复骨缺损区,B组较C组修复明显。X线评分结果：A组在各时期均明显高于B组,差异具有显著性（P〈0．01）。结论：自体BMSCs与β-TCP／CPPF／PLLA复合物移植能修复大节段的骨干缺损。     

The effect of bioengineered acellular collagen patch on cardiac remodeling and ventricular function post myocardial infarction

Regeneration of the damaged myocardium is one of the most challenging fronts in the field of tissue engineering due to the limited capacity of adult heart tissue to heal and to the mechanical and structural constraints of the cardiac tissue. In this study we demonstrate that an engineered acellular scaffold comprising type I collagen, endowed with specific physiomechanical properties, improves cardiac function when used as a cardiac patch following myocardial infarction. Patches were grafted onto the infarcted myocardium in adult murine hearts immediately after ligation of left anterior descending artery and the physiological outcomes were monitored by echocardiography, and by hemodynamic and histological analyses four weeks post infarction. In comparison to infarcted hearts with no treatment, hearts bearing patches preserved contractility and significantly protected the cardiac tissue from injury at the anatomical and functional levels. This improvement was accompanied by attenuated left ventricular remodeling, diminished fibrosis, and formation of a network of interconnected blood vessels within the infarct. Histological and immunostaining confirmed integration of the patch with native cardiac cells including fibroblasts, smooth muscle cells, epicardial cells, and immature cardiomyocytes. In summary, an acellular biomaterial with specific biomechanical properties promotes the endogenous capacity of the infarcted myocardium to attenuate remodeling and improve heart function following myocardial infarction.     

Synthesis and Evaluation of Long-Acting D-Penicillamine Derivatives

This study extends the use of two lathyrogens, β-aminopropionitrile (BAPN) and D-penicillamine (DPA) from daily systemic or local-topical administration to long-time acting agents. This was achieved by converting the hydrophilic drugs into lipophilic derivatives. The synthesis of functional derivatives of DPA consisted in esterification with methyl-, hexyl-, or benzyl alcohols in the presence of thionylchloride. The esters formed were hydrochlorides, acidic and soluble in water. During neutralization in vitro or in vivo by tissue fluid, an oily substance is formed that elutes from a hydrogel polymer at a much slower rate than hydroplilic DPA itself. The degree of lipophilicity, measured as a partition coefficient between octanol/water, was highest for hexyl ester and lowest for methyl ester DPA. A single injection of either DPA hexyl ester HCl or 3-hexyl(amino) propionitrile into the full thickness skin incision wound in rats significantly lowered the breaking strength of the wound 12 days after injection, indicating the interference with collagen cross-linking. Both agents injected into the breast adenocarcinoma in Fisher rats significantly inhibited tumor growth without any signs of local or systemic toxicity. We conclude that these lipophilic lathyrogens with prolonged effectiveness are suitable in the treatment of pathologies, consisting of excessively cross-linked or deposited collagen (fibrotic adhesions, strictures, stenosis, and scar contractures) and in the treatment of single, solitary tumors, malignant and benign.     

Effect of bioactive borate glass microstructure on bone regeneration,angiogenesis, and hydroxyapatite conversion in a rat calvarial defect model

Borate bioactive glasses are biocompatible and enhance new bone formation, but the effect of their microstructure on bone regeneration has received little attention. In this study scaffolds of borate bioactive glass (1393B3) with three different microstructures (trabecular, fibrous, and oriented) were compared for their capacity to regenerate bone in a rat calvarial defect model. 12 weeks post-implantation the amount of new bone, mineralization, and blood vessel area in the scaffolds were evaluated using histomorphometric analysis and scanning electron microscopy. The amount of new bone formed was 33%, 23%, and 15%, respectively, of the total defect area for the trabecular, oriented, and fibrous microstructures. In comparison, the percent new bone formed in implants composed of silicate 45S5 bioactive glass particles (250–300 μm) was 19%. Doping the borate glass with copper (0.4 wt.% CuO) had little effect on bone regeneration in the trabecular and oriented scaffolds, but significantly enhanced bone regeneration in the fibrous scaffolds (from 15 to 33%). The scaffolds were completely converted to hydroxyapatite within the 12 week implantation. The amount of hydroxyapatite formed, 22%, 35%, and 48%, respectively, for the trabecular, oriented, and fibrous scaffolds, increased with increasing volume fraction of glass in the as-fabricated scaffold. Blood vessels infiltrated into all the scaffolds, but the trabecular scaffolds had a higher average blood vessel area compared with the oriented and fibrous scaffolds. While all three scaffold microstructures were effective in supporting bone regeneration, the trabecular scaffolds supported more bone formation and may be more promising in bone repair.     

A mechanism for effective cell-seeding in rigid,microporous substrates

Seeding cells into porous ceramic substrates has been shown to improve outcomes in surgical repair of large bone defects, but the physics underlying cellular ingress into such scaffolds remains elusive. This paper demonstrates capillary forces as a novel, yet simple, self-loading or self-seeding mechanism for rigid, microporous substrates. Capillary forces were found to draw cells through a microporous network with interconnections smaller than the diameter of the cells in suspension. Work here emphasizes CaP-based bone scaffolds containing both macroporosity (>100 μm) and microporosity (5–50 μm); these have been shown to improve bone formation in vivo as compared to their macroporous counterparts and also performed better than microporous scaffolds containing BMP-2 by some measures of bone regeneration. We hypothesize that capillary force driven self-seeding in both macro- and micropores may underlie this improvement, and present a mathematical model and experiments that support this hypothesis. The cell localization and penetration depth within these two-dimensional substrates in vitro depends upon both the cell type (size and stiffness) and the capillary forces generated by the microstructure. Additional experiments showing that cell penetration depth in vitro depends on cell size and stiffness suggest that microporosity could be tailored to optimize cell infiltration in a cell-specific way. Endogenous cells are also drawn into the microporous network in vivo. Results have important implications for design of scaffolds for the healing of large bone defects, and for controlled release of drugs in vivo.     

Combining self-assembling peptide gels with three-dimensional elastomer scaffolds

Some of the problems raised by the combination of porous scaffolds and self-assembling peptide (SAP) gels as constructs for tissue engineering applications are addressed for the first time. Scaffolds of poly(ethyl acrylate) and the SAP gel RAD16-I were employed. The in situ gelation of the SAP gel inside the pores of the scaffolds was studied. The scaffold-cum-gel constructs were characterized morphologically, physicochemically and mechanically. The possibility of incorporating an active molecule (bovine serum albumin, taken here as a model molecule for others) in the gel within the scaffold’s pores was assessed, and the kinetics of its release in phosphate-buffered saline was followed. Cell seeding and colonization of these constructs were preliminarily studied with L929 fibroblasts and subsequently checked with sheep adipose-tissue-derived stem cells intended for further preclinical studies. Static (conventional) and dynamically assisted seedings were compared for bare scaffolds and the scaffold-cum-gel constructs. The SAP gel inside the pores of the scaffold significantly improved the uniformity and density of cell colonization of the three-dimensional (3-D) structure. These constructs could be of use in different advanced tissue engineering applications, where, apart from a cell-friendly extracellular matrix -like aqueous environment, a larger-scale 3-D structure able to keep the cells in a specific place, give mechanical support and/or conduct spatially the tissue growth could be required.